Although the underlying mechanism leading to tissue fibrosis is not entirely clear, the hallmark of a fibrotic response is the excessive accumulation of collagen resulting from enhanced fibroblast proliferation and collagen production. The most specific way to interfere with collagen overproduction would be at the level of post-translational modifications unique to collagen biosynthesis. Two such modifications that have received considerable attention as potential sites for pharmacological intervention are the intracellular hydroxylation of susceptible proline residues, catalyzed by prolyl hydroxylase, and the extracellular oxidation of selected lysine residues, catalyzed by lysyl oxidase.
The present invention provides a novel pharmacological approach to interfere with collagen biosynthesis involving lysyl hydroxylase as the target enzyme. Catalyzing the synthesis of hydroxylysine during collagen biosynthesis, this post-translational modifying enzyme is critically important in the formation of intermolecular crosslinks that stabilize the fibrillar structure of collagen. Its activity is increased during lung fibrosis, leading to enrichment of hydroxylysyl crosslinks in newly synthesized collagen. Inhibition of lysyl hydroxylase would result in the synthesis of a collagen deficient in hydroxylysine, and, therefore, hydroxylysine-derived crosslinks. Such a collagen would be deposited in the extracellular matrix as a non-functional protein and would also be more susceptible to degradation by collagenase, limiting its amount in the fibrotic tissue.
Inhibition of crosslinking at the level of lysyl hydroxylation has a distinct advantage in that it would lead to inhibition of collagen biosynthesis without affecting the biosynthesis of elastin which lacks hydroxylysine, thus avoiding cardiovascular side-effects seen with crosslink inhibitors that act at the level of lysyl oxidation or subsequent condensation.
Minoxidil has been identified as a pharmacological agent that has the unique ability to selectively decreased the activity of lysyl hydroxylase in cultured fibroblasts, apparently by inhibiting enzyme synthesis at the transcriptional level. (See Murad and Pinnell, J. Biol. Chem. 262:11,973 (1987), see also generally Hautala et al, Biochem. J. 283:51 (1992)). In addition, minoxidil is capable of inhibiting the serum-induced proliferation of fibroblasts in culture, a property that would serve to further reduce the amount of collagen in the fibrotic tissue. However, the hypotensive activity of minoxidil is a deterrent in its use as an antifibrotic drug in normotensive patients.
The present invention results, at least in part, from the discovery that two derivatives of minoxidil, 3'-hydroxyminoxidil and 4'-hydroxyminoxidil (see FIG. 1; see also U.S. Pat. No. 3,998,827), are inactive as antihypertensive agents, but are as active as minoxidil in suppressing fibroblast proliferation and lysyl hydroxylase activity. The hydroxy derivatives of minoxidil are also more water soluble than the parent compound, are not cytotoxic even at a high concentration (5 mM), and retain the enzyme suppressing activity at this concentration, at which minoxidil mysteriously loses its effect. The hydroxyminoxidils are thus preferred candidates for antifibrotic drugs.